Microarrays and uses therefor

ABSTRACT

Methods of using microarrays to simplify analysis and characterization of genes and their function are provided. Such methods can be used to identify and characterize antibodies having binding affinity for a specific target antigen. A method of determining gene expression at the protein level by contacting an array of characterized or uncharacterized antibodies on a solid surface with one or more proteins and identifying the antibodies to which said protein(s) binds also is provided. This method can be used to compare the protein expression in two different populations of cells, such as normal cells and cancer cells or resting cells and stimulated cells. In addition, a method of determining gene expression at the protein level by contacting a microarray of nucleic acid samples derived from a variety of different sources with one or more nucleic acid probes then identifying the sample or samples to which the probe binds is provided.

This application is a divisional application of U.S. Ser. No.09/245,615, filed Feb. 4, 1999, which claims the benefit of priorityunder 35 U.S.C. §119 of U.S. Ser. No. 60/073,605, filed Feb. 4, 1998(now abandoned), the entire contents of each of which is incorporatedherein by reference.

FIELD OF THE INVENTION

The invention disclosed herein relates to new methods of usingmicroarray technologies. The methods are useful for identifying andcharacterizing specific antibodies as well as the characterization ofdifferent tissues or cells by protein or nucleic acid analysis.

BACKGROUND OF THE INVENTION

Recent breakthroughs in nucleic acid sequencing technology have madepossible the sequencing of entire genomes from a variety of organisms,including humans. The potential benefits of a complete genome sequenceare many, ranging from applications in medicine to a greaterunderstanding of evolutionary processes. These benefits cannot be fullyrealized, however, without an understanding of how and where these newlysequenced genes function.

Traditionally, functional understanding started with recognizing anactivity, isolating a protein associated with that activity, thenisolating the gene, or genes, encoding that protein. The isolatedprotein was also used to generate antibody reagents. Specific antibodiesand fragments of the isolated gene were both employed to study tissueexpression and function.

Several methods have been used to study protein expression patternsincluding in situ hybridization studies of tissue sections and northernblots. These methods are both time consuming and require relativelylarge amounts of material to perform successfully.

Antibodies that bind to specific antigens have been produced by avariety of methods including immunization of animals, fusion ofmammalian spleen cells to immortalized cells to produce hybridomas,random peptide generation using phage or bacterial display andconstrained peptide libraries. Regardless of how the desired antibody isgenerated, the methods currently available to identify one with aparticular binding specificity are generally laborious and incapable ofthe simultaneous testing of large numbers of unknowns.

One method involves binding the antigen to a porous membrane, such asnitrocellulose, contacting the membrane with a source of testantibodies, then determining whether or not any of the test antibodieshas bound to the antigen. This method only allows the testing of onesource of test antibodies per piece of porous membrane, making themethod both inconvenient and wasteful of materials.

Antibody/antigen reactions can also be evaluated in plastic plates, suchas 96-well microtiter plates, using methods similar to those describedabove. This method is likewise limited in the number of samples that canbe tested in any one assay, thus requiring many assays to fully evaluatea large number of antibody unknowns. Chang (U.S. Pat. No. 4,591,570,issued May 27, 1986) describes an array of a limited number ofcharacterized antibodies to known antigens on a glass surface that canbe used to bind to specific antigens on the surface of whole cells.

Recently new technologies have arisen that allow the creation ofmicroarrays containing thousands or millions of different elements. Sucharray technology has been applied mainly to forming arrays of individualnucleic acids (see, for example, Marshall and Hodgson, Nature Biotech.16:27-31, 1998; Ramsay, Nature Biotech. 16:40-44, 1998), in particularshort oligonucleotides synthesized in situ.

Methods are needed to simply and rapidly screen very large numbers ofuncharacterized antibodies for those specific for a given antigen aswell as for the characterization of tissues and cells by nucleic acidand/or protein analysis. The invention described herein addresses thatneed.

BRIEF DESCRIPTION OF THE INVENTION

The invention disclosed herein comprises methods of using microarrays tosimplify analysis and characterization of genes and their function. Inone aspect of the invention the methods are used to identify andcharacterize antibodies having binding affinity for a specific targetantigen. This method comprises contacting an array of uncharacterizedantibodies bound to a solid surface with at least one target antigen andidentifying the antibodies to which the target antigen binds. The methodcan be performed under a variety of conditions to identify antibodieswith a range of binding affinities.

A second aspect of the invention comprises a method of determining geneexpression at the protein level comprising contacting an array ofcharacterized or uncharacterized antibodies on a solid surface with oneor more proteins and identifying the antibodies to which said protein(s)binds. This method can be further used to compare the protein expressionin two different populations of cells, such as normal cells and cancercells or resting cells and stimulated cells. A related embodiment can beused as a tool in the diagnosis of various disorders.

A further aspect of the invention comprises a method of determining geneexpression at the protein level comprising contacting a microarray ofnucleic acid samples derived from a variety of different sources withone or more nucleic acid probes then identifying the sample or samplesto which the probe binds.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, and 1C show microarrays of antibodies bound to positivelycharged nylon, reacted with antigen and detected by non-fluorescentmeans.

FIG. 2 shows a microarray produced using a robotic arraying apparatus.Antigen binding is detected by non-fluorescent means.

FIG. 3 shows the ability of the antibody microarrays to evaluaterelative binding affinities to a specific antigen.

FIG. 4 shows a microarray of polyclonal antibodies in comparison to amicroarray of monoclonal antibodies.

FIGS. 5A, 5B and 5C show a microarray of antibodies reacted with a celllysate under conditions that vary the amount of background binding.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses methods of using microarrays to simplifyanalysis and characterization of genes and their function. In a firstaspect of the invention the methods are used for identifying andcharacterizing antibodies having binding specificity to a particularantigen or set of antigens. This method utilizes microarray technologyto create ordered matrices of large numbers of uncharacterizedantibodies which can then be contacted with antigen under a variety ofconditions. The method is rapid and simple to perform and is applicableto the simultaneous screening of very large numbers of antibodies.

Briefly, uncharacterized antibodies are bound to a solid surface in anarray format consisting of discrete spots whose spatial location can beeasily identified. Each location represents an antibody from a knownsource, such as a particular hybridoma growing in a well in a 96-wellmicrotiter plate. The space between the antibody spots is treated tominimize non-specific binding to the solid support. The arrayedantibodies are then contacted with an antigen, or a set of antigens, forwhich specific antibodies are sought. The antigen solution is left incontact with the array for an amount of time sufficient to allowantigen:antibody complexes to form (generally 10 minutes to 2 hours),then the unbound antigen is washed away under suitable conditions. Boundantigen is detected at a particular antibody spot using one of a varietyof detection methods, thus identifying the source of an antibodyspecific for the particular antigen.

The term “antibody” is used herein in the broadest sense andspecifically includes intact monoclonal antibodies, polyclonalantibodies, multispecific antibodies (e.g. bispecific antibodies) formedfrom at least two intact antibodies, and antibody fragments, includingsingle chain antibodies, so long as they exhibit the desired bindingproperties as described herein.

Various procedures well-known in the art may be used for the productionof polyclonal antibodies to an epitope or antigen of interest. A hostanimal of any of a number of species, such as rabbits, goats, sheep,horse, cow, mice, rats, etc. is immunized by injection with an antigenicpreparation which may be derived from cells or microorganisms, or may berecombinantly or synthetically produced. Various adjuvants well known inthe art may be used to enhance the production of antibodies by theimmunized host, for example, Freund's adjuvant (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanins, dinitrophenol, liposomes,potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin)and Propionibacterium acanes, and the like.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. Preferred antibodies are mAbs, which may be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, and any subclass orisotype thereof.

In addition to their specificity, monoclonal antibodies are advantageousin that they are synthesized by hybridoma culture, uncontaminated byother immunoglobulins. The modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by the hybridoma method first described by Kohleret al., Nature, 256:495 (1975), or may be made by recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567, incorporated by referenceherein). The “monoclonal antibodies” may also be isolated from phageantibody libraries using the techniques described in Clackson et al.,Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597(1991), for example.

The monoclonal antibodies contemplated for use herein specificallyinclude “chimeric” antibodies (immunoglobulins) in which a portion ofthe heavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, so long as they exhibit the desired biological activity(U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from acomplementarity-determining region (CDR) of the recipient are replacedby residues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity, andcapacity. In some instances, Fv framework region (FR) residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. These modifications are made to further refine and maximizeantibody performance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin sequence. The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature,321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol., 2:593-596 (1992). The humanizedantibody includes a PRIMATIZED™ antibody wherein the antigen-bindingregion of the antibody is derived from an antibody produced byimmunizing macaque monkeys with the antigen of interest.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al. Protein Eng.8(10):1057-1062 (1995)); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

Particularly preferred in the practice of the invention are single-chainantibodies. “Single-chain” or “sFv” antibodies are antibody fragmentscomprising the V_(H) and V_(L) domains of an antibody, wherein thesedomains are present in a single polypeptide chain. Preferably, the Fvpolypeptide further comprises a polypeptide linker between the V_(H) andV_(L) domains which enables the sFv to form the desired structure forantigen binding. For a review of sFvs see Pluckthun in The Pharmacologyof Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,Springer-Verlag, New York, pp. 269-315 (1994).

Large quantities of single chain antibodies with uncharacterizedrandomized binding specificity can be produced using a number ofmethodologies known in the art. Recombinant antibody libraries can becreated in filamentous phage particles (Daniels and Lane, Methods9(3):494-507, 1996; Reichmann and Weill, Biochemistry 32(34):8848-8855;Rader and Barbas, Curr Opin Biotechnol 9(4):503-508, 1997; Iba andKurosawa, Immunol Cell Biol 75(2):217-221, 1997, WO 90/05144, WO92/01047, WO 92/20791, WO 93/19172, GB 9722131.8, GB9810228.8 and GB9810223.9, all of which are incorporated by reference herein in theirentirety), for example, or similarly in yeast, bacteria, and the like.Other methods for creating random libraries of sFvs include varioussolid state synthesis methods.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

The antibodies employed in the invention can be isolated prior tocreating a microarray. An “isolated” molecule, whether an antibody,antigen or nucleic acid, is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with particular uses for the molecule, and may includeenzymes, hormones, and other proteinaceous or nonproteinaceous solutes.In preferred embodiments, a protein will be purified (1) to greater than95% by weight of protein as determined by the Lowry method, and mostpreferably more than 99% by weight, (2) to a degree sufficient to obtainat least 15 residues of N-terminal or internal amino acid sequence byuse of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGEunder reducing or nonreducing conditions using Coomassie blue or,preferably, silver stain. Isolated protein includes the protein in situwithin recombinant cells since at least one component of the protein'snatural environment will not be present. Ordinarily, however, isolatedprotein will be prepared by at least one purification step. Unpurifiedantibodies, such as those found in serum, can also be employed in thepresent invention.

By “isolated” in reference to nucleic acid is meant a polymer of 14, 17,21 or more contiguous nucleotides, including DNA or RNA that is isolatedfrom a natural source or that is synthesized. The isolated nucleic acidof the present invention is unique in the sense that it is not found ina pure or separated state in nature. Use of the term “isolated”indicates that a naturally occurring sequence has been removed from itsnormal cellular (i.e., chromosomal) environment. Thus, the sequence maybe in a cell-free solution or placed in a different cellularenvironment. The term does not imply that the sequence is the onlynucleotide sequence present, but that it is essentially free (about90-95% pure at least) of non-nucleotide material naturally associatedwith it and thus is meant to be distinguished from isolated chromosomes.

One particularly useful method of isolating antibodies, such as singlechain antibodies from a cell extract, is affinity purification. Resinssuitable for antibody purification are well known in the art, forexample, protein A SEPHAROSE™. A recombinant antibody can be engineeredto contain an affinity purification tag to facilitate its purification.Resins suitable for antibody purification are well known in the art, forexample, protein A SEPHAROSE™ resin.

Affinity purification tags are generally peptide sequences that caninteract with a binding partner immobilized on a solid support.Synthetic DNA sequences encoding multiple consecutive single aminoacids, such as histidine, when fused to the expressed protein, may beused for one-step purification of the recombinant protein by highaffinity binding to a resin column, such as nickel SEPHAROSE™ resin. Anendopeptidase recognition sequence can be engineered between thepolyamino acid tag and the protein of interest to allow subsequentremoval of the leader peptide by digestion with enterokinase, and otherproteases. Sequences encoding peptides such as the chitin binding domain(which binds to chitin), biotin (which binds to avidin and strepavidin),and the like can also be used for facilitating purification of theprotein of interest. The affinity purification tag can be separated fromthe protein of interest by methods well known in the art, including theuse of inteins (protein self-splicing elements, Chong, et al, Gene192:271-281, 1997).

By using an amount of resin with binding sites sufficient for only asmall portion of the antibody present in the unpurified mixture, theprocess of isolation can be used to simultaneously normalize yield andisolate the antibody. For example, although each sample will contain adifferent and unknown amount of antibody protein, the samples can becontacted with an amount of resin whose maximum binding capacity is 10mgs. Thus any antibody greater than this amount will pass through theresin unbound. The maximum bound amount can then be eluted from theresin.

Methods for creating microarrays are known in the art including printingon a solid surface using pins (passive pins, quill pins, and the like)or spotting with individual drops of solution. Passive pins draw upenough sample to dispense a single spot. Quill pins draw up enoughliquid to dispense multiple spots. Bubble printers use a loop to capturea small volume which is dispensed by pushing a rod through the loop.Microdispensing uses a syringe mechanism to deliver multiple spots of afixed volume. In addition, solid supports, can be arrayed usingpiezoelectric (ink jet) technology, which actively transfers samples toa solid support.

One method is described in Shalon and Brown (WO 95/35505, published Dec.28, 1995) which is incorporated herein by reference in its entirety. Themethod and apparatus described in Shalon and Brown can create an arrayof up to six hundred spots per square centimeter on a glass slide usinga volume of 0.01 to 100 nl per spot. Suitable concentrations of antibodyrange from about 1 ng/μl to about 1 μg/μl. In the present invention,each spot can contain one or more than one distinct antibody.

Other methods of creating arrays are known in the art, includingphotolithographic printing (Pease, et al, Proc. Natl. Acad. Sci. USA,91(11):5022-5026, 1994) and in situ synthesis. While known in situsynthesis methods are less useful for synthesizing polypeptides longenough to be antibodies, they can be used to make polypeptides up to 50amino acids in length, which can serve as binding proteins as describedbelow.

The microarrays can be created on a variety of solid surfaces such asplastics (e.g. polycarbonate), complex carbohydrates (e.g. agarose andSEPHAROSE™), acrylic resins (e.g. polyacrylamide and latex beads), andnitrocellulose. Preferred solid support materials include glass slides,silicon wafers, and positively charged nylon. Specific examples ofsuitable solid supports are described in the Examples below.

Methods for covalent attachment of antibodies to a solid support areknown in the art. Examples of such methods are found in Bhatia, et al,Anal. Biochem. 178(2):408-413, 1989; Ahluwalia, et al, Biosens.Bioelectron. 7(3):207-214, 1992; Jonsson, et al, Biochem. J.227(2):373-378, 1985; and Freij-Larsson, et al, Biomaterials17(22):2199-2207, 1996, all of which are incorporated by referenceherein in their entirety. Proteins may additionally be attached to asolid support using methods described in the examples below.

Methods of reducing non-specific binding to a solid surface are wellknown in the art and include washing the arrayed solid surface withbovine serum albumin (BSA), reconstituted non-fat milk, salmon spermDNA, porcine heparin, and the like (see Ausubel, et al., Short Protocolsin Molecular Biology, 3rd ed. 1995).

The arrays used to identify antigen-specific antibodies are contactedwith a solution containing one or more known antigens in order toidentify antibodies in the array with binding specificity for theantigen. The antigens are often proteins, although they may also beorganic chemical compounds, carbohydrates, nucleic acids, and the like.They may be isolated or semi-isolated, recombinant or naturallyoccurring. The amount of antigen used can vary from about 1-100 ng/μl.The antigen is left in contact with the array for an amount of timesufficient for antibody:antigen complexes to form, should one of theantibodies in the array be specific for the antigen. The amount of timesufficient for this purpose will range from 5 minutes to 24 hours, andwill generally be from 0.5 to 2 hours.

One antigen of particular interest in the practice of the invention isrecombinant protein, either a full-length gene product or a fragmentthereof, for example an Expressed Sequence Tag (or EST fragment). ESTfragments are relatively short cDNA sequences that have been randomlygenerated and sequenced, generally as part of an ongoing effort to mapan entire genome (Adams, et al, Science 252(5013):1651-1656, 1991).Large numbers of these sequences are available in public databases. Theidentity of the proteins encoded by the vast majority of these sequencesis unknown. The following discussion, although directed to theexpression of EST-encoded peptides, is equally applicable to anyexpressed product of a nucleic acid sequence, including full-lengthproteins.

Techniques are available in the art by which cells can be geneticallyengineered to express the peptide encoded by a given EST fragment. Themethods of the invention can then be used to identify antibodiesspecific for the peptide. These antibodies are then useful as reagentsthat can be employed in purification and identification of thefull-length protein, and in other experimental procedures designed toelucidate the protein's location and function.

Prokaryotic hosts are, generally, very efficient and convenient for theproduction of recombinant proteins and are, therefore, one type ofpreferred expression system for EST fragments. Prokaryotes mostfrequently are represented by various strains of E. coli. However, othermicrobial strains may also be used, including other bacterial strains.

In prokaryotic systems, plasmid vectors that contain replication sitesand control sequences derived from a species compatible with the hostmay be used. Examples of suitable plasmid vectors may include pBR322,pUC118, pUC119, and the like; suitable phage or bacteriophage vectorsmay include λgt10, λgt11, and the like; and suitable virus vectors mayinclude pMAM-neo, PKRC and the like. Preferably, the selected vector ofthe present invention has the capacity to replicate in the selected hostcell.

Recognized prokaryotic hosts include bacteria such as E. coli and thosefrom genera such as Bacillus, Streptomyces, Pseudomonas, Salmonella,Serratia, and the like. However, under such conditions, the polypeptidewill not be glycosylated. The prokaryotic host selected for use hereinmust be compatible with the replicon and control sequences in theexpression plasmid.

To express an EST fragment in a prokaryotic cell, it is necessary tooperably link the gene sequence to a functional prokaryotic promotersuch as the T7 promoter or RSC promoter. Such promoters may be eitherconstitutive or, more preferably, regulatable (i.e., inducible orderepressible). Examples of constitutive promoters include the intpromoter of bacteriophage λ, the bla promoter of the β-lactamase genesequence of pBR322, the CAT promoter of the chloramphenicol acetyltransferase gene sequence of pPR325, and the like. Examples of inducibleprokaryotic promoters include the major right and left promoters ofbacteriophage (P_(L) and P_(R)), the trp, reca, lacZ, LacI, and galpromoters of E. coli, the α-amylase (Ulmanen et al., J. Bacteriol.162:176-182, 1985) and the sigma-28-specific promoters of B. subtilis(Gilman et al., Gene sequence 32:11-20(1984)), the promoters of thebacteriophages of Bacillus (Gryczan, In: The Molecular Biology of theBacilli, Academic Press, Inc., NY (1982)), Streptomyces promoters (Wardet at., Mol. Gen. Genet. 203:468-478, 1986), and the like. Exemplaryprokaryotic promoters are reviewed by Glick (J. Ind. Microbiol.1:277-282, 1987); Cenatiempo (Biochimie 68:505-516, 1986); and Gottesman(Ann. Rev. Genet. 18:415-442, 1984).

Proper expression in a prokaryotic cell also requires the presence of aribosome binding site upstream of the gene sequence-encoding sequence.Such ribosome binding sites are disclosed, for example, by Gold et at.(Ann. Rev. Microbiol. 35:365-404, 1981). The selection of controlsequences, expression vectors, transformation methods, and the like, aredependent on the type of host cell used to express the gene.

Host cells which may be used in the expression systems of the presentinvention are not strictly limited, provided that they are suitable foruse in the expression of the peptide of interest. Suitable hosts mayoften include eukaryotic cells. Preferred eukaryotic hosts include, forexample, yeast, fungi, insect cells, and mammalian cells either in vivo,or in tissue culture. Mammalian cells which may be useful as hostsinclude HeLa cells, cells of fibroblast origin such as VERO, 3T3 orCHOK1, HEK 293 cells or cells of lymphoid origin (such as 32D cells) andtheir derivatives. Preferred mammalian host cells include SP2/0 andJS58L, as well as neuroblastoma cell lines such as IMR 332 and PC12which may provide better capacities for correct post-translationalprocessing.

In addition, plant cells are also available as hosts, and controlsequences compatible with plant cells are available, such as thecauliflower mosaic virus 35S and 19S, nopaline synthase promoter andpolyadenylation signal sequences, and the like. Another preferred hostis an insect cell, for example the Drosophila larvae. Using insect cellsas hosts, the Drosophila alcohol dehydrogenase promoter can be used.Rubin, Science 240:1453-1459, 1988). Alternatively, baculovirus vectorscan be engineered to express large amounts of peptide encoded by an ESTfragment in insects cells (Jasny, Science 238:1653, 1987); Miller etal., In: Genetic Engineering (1986), Setlow, J. K., et al., eds.,Plenum, Vol. 8, pp. 277-297).

Any of a series of yeast gene sequence expression systems can beutilized which incorporate promoter and termination elements from theactively expressed gene sequences coding for glycolytic enzymes whichare produced in large quantities when yeast are grown in media rich inglucose. Known glycolytic gene sequences can also provide very efficienttranscriptional control signals. Yeast provides substantial advantagesin that it can also carry out post-translational peptide modifications.A number of recombinant DNA strategies exist which utilize strongpromoter sequences and high copy number of plasmids which can beutilized for production of the desired proteins in yeast. Yeastrecognizes leader sequences on cloned mammalian gene sequence productsand secretes peptides bearing leader sequences (i.e., pre-peptides). Fora mammalian host, several possible vector systems are available for theexpression of and EST fragment.

A wide variety of transcriptional and translational regulatory sequencesmay be employed, depending upon the nature of the host. Thetranscriptional and translational regulatory signals may be derived fromviral sources, such as adenovirus, bovine papilloma virus,cytomegalovirus, simian virus, or the like, where the regulatory signalsare associated with a particular gene sequence which has a high level ofexpression. Alternatively, promoters from mammalian expression products,such as actin, collagen, myosin, and the like, may be employed.Transcriptional initiation regulatory signals may be selected whichallow for repression or activation, so that expression of the genesequences can be modulated. Of interest are regulatory signals which aretemperature-sensitive so that by varying the temperature, expression canbe repressed or initiated, or are subject to chemical (such asmetabolite) regulation.

Expression of an EST fragment in eukaryotic hosts involves the use ofeukaryotic regulatory regions. Such regions will, in general, include apromoter region sufficient to direct the initiation of RNA synthesis.Preferred eukaryotic promoters include, for example, the promoter of themouse metallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gen.1:273-288, 1982); the TK promoter of Herpes virus (McKnight, Cell31:355-365, 1982); the SV40 early promoter (Benoist et al., Nature(London) 290:304-310, 1981); the yeast ga14 gene sequence promoter(Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975, 1982);Silver et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955, 1984), the CMVpromoter, the EF-1 promoter, and the like.

An EST fragment and an operably linked promoter may be introduced into arecipient prokaryotic or eukaryotic cell either as a nonreplicating DNA(or RNA) molecule, which may either be a linear molecule or, morepreferably, a closed covalent circular molecule (a plasmid). Since suchmolecules are incapable of autonomous replication, the expression of thegene may occur through the transient expression of the introducedsequence. Alternatively, permanent or stable expression may occurthrough the integration of the introduced DNA sequence into the hostchromosome.

A vector may be employed which is capable of integrating the desiredgene sequences into the host cell chromosome. Cells which have stablyintegrated the introduced DNA into their chromosomes can be selected byalso introducing one or more markers which allow for selection of hostcells which contain the expression vector. The marker may provide forprototrophy to an auxotrophic host, biocide resistance, e.g.,antibiotics, or heavy metals, such as copper, or the like. Theselectable marker gene sequence can either be directly linked to the DNAgene sequences to be expressed, or introduced into the same cell bycotransfection. Common selectable marker gene sequences include thosefor resistance to antibiotics such as ampicillin, tetracycline,kanamycin, bleomycin, streptomycin, hygromycin, neomycin, Zeocin™, andthe like. Selectable auxotrophic gene sequences include, for example,hisD, which allows growth in histidine free media in the presence ofhistidinol.

Additional elements may also be needed for optimal synthesis of singlechain binding protein mRNA. These elements may include splice signals,as well as transcription promoters, enhancers, and termination signals.cDNA expression vectors incorporating such elements include thosedescribed by Okayama, Mol. Cell. Bio. 3:280, 1983.

The recombinant antigen may be produced as a fusion protein. When twoprotein-coding sequences not normally associated with each other innature are in the same reading frame the resulting expressed protein iscalled a “fusion protein” as two distinct proteins have been “fused”together. Fusion proteins have a wide variety of uses. For example, twofunctional enzymes can be fused to produce a single protein withmultiple enzymatic activities or short peptide sequences, such asepitope tags or affinity purification tags (see above), can be fused toa larger protein and serve as aids in purification or as means ofidentifying the expressed protein by serving as epitopes detectable byspecific antibodies.

Epitope tags are short peptide sequences that are recognized byepitope-specific antibodies. A fusion protein comprising a recombinantprotein and an epitope tag can be simply and easily purified using anantibody bound to a chromatography resin. The presence of the epitopetag furthermore allows the recombinant protein to be detected insubsequent assays, such as Western blots, without having to produce anantibody specific for the recombinant protein itself. Examples ofcommonly used epitope tags include V5, glutathione-S-transferase (GST),hemagglutinin (HA), the peptide Phe-His-His-Thr-Thr, chitin bindingdomain, and the like.

A fusion protein may be a means by which the recombinant antigen proteincan be easily detected. For example, the fusion component can itself bea detectable moiety, such as fluorescent protein (fluorescent greenprotein, fluorescent yellow protein, and the like), or alternatively canbe one member of a specific binding pair (such as biotin andstreptavidin, for example) which can be detected by reacting with theother member conjugated to a detectable substance.

The foregoing elements can be combined to produce vectors suitable foruse in the methods of the invention. Those of skill in the art would beable to select and combine the elements suitable for use in theirparticular system.

The introduced nucleic acid molecule can be incorporated into a plasmidor viral vector capable of autonomous replication in the recipient host.Any of a wide variety of vectors may be employed for this purpose.Factors of importance in selecting a particular plasmid or viral vectorinclude: the ease with which recipient cells that contain the vector maybe recognized and selected from those recipient cells which do notcontain the vector; the number of copies of the vector which are desiredin a particular host; and whether it is desirable to be able to“shuttle” the vector between host cells of different species.

Suitable prokaryotic vectors include plasmids such as those capable ofreplication in E. coli (for example, pBR322, Co1E1, pSC101, PACYC 184,itVX, pRSET, pBAD (Invitrogen, Carlsbad, Calif.), and the like). Suchplasmids are disclosed by Sambrook (cf. “Molecular Cloning: A LaboratoryManual”, second edition, edited by Sambrook, Fritsch, & Maniatis, ColdSpring Harbor Laboratory, (1989)). Bacillus plasmids include pC194,pC221, pT127, and the like, and are disclosed by Gryczan (In: TheMolecular Biology of the Bacilli, Academic Press, NY (1982), pp.307-329). Suitable Streptomyces plasmids include p1J101 (Kendall et al.,J. Bacteriol. 169:4177-4183, 1987), and Streptomyces bacteriophages suchas φC31 (Chater et al., In: Sixth International Symposium onActinomycetales Biology, Akademiai Kaido, Budapest, Hungary (1986), pp.45-54). Pseudomonas plasmids are reviewed by John et al. (Rev. Infect.Dis. 8:693-704, 1986), and Izaki (Jpn. J. Bacteriol. 33:729-742, 1978).

Suitable eukaryotic plasmids include, for example, BPV, vaccinia, SV40,2-micron circle, pCDN3.1 (Invitrogen), and the like, or theirderivatives. Such plasmids are well known in the art (Botstein et al.,Miami Wntr. Symp. 19:265-274, 1982); Broach, In: “The Molecular Biologyof the Yeast Saccharomyces: Life Cycle and Inheritance”, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., p. 445-470 (1981); Broach,Cell 28:203-204, 1982); Dilon et at., J. Clin. Hematol. Oncol. 10:39-48,1980); Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3,Gene Sequence Expression, Academic Press, NY, pp. 563-608 (1980).

Once antibody:antigen complexes have been formed and unbound antigenwashed away under suitable conditions, the antibody:antigen complexescan be detected using one of several techniques known in the art.Suitable washing conditions are known to those skilled in the art (see,for example, Ausubel, et al, Short Protocols in Molecular Biology, 3rded. 1995). Exemplary washing conditions are shown in the examples below.

For detection in the case of recombinant antigens, expression vectorscan be used that form chimeric fusion peptides as described above. Theepitope tagged antigen can be detected using an antibody specific forthe tag sequence. This antibody may be itself detectably labeled or canbe detected with a third detectably-labeled antibody. Alternatively, theantigen can be complexed with biotin and detected usingdetectably-labeled avidin or streptavidin. The antigen itself can alsobe detectably labeled, such as with a fluorescent dye compound.

The term “detectably labeled” as used herein is intended to encompassantigen directly coupled to a detectable substance, such as afluorescent dye, and antigen coupled to a member of binding pair, suchas biotin/streptavidin, or an epitope tag that can specifically interactwith a molecule that can be detected, such as by producing a coloredsubstrate or fluorescence.

Substances suitable for detectably labeling proteins include fluorescentdyes such as fluorescein isothiocyanate (FITC), fluorescein, rhodamine,tetramethyl-rhodamine-5-(and 6)-isothiocyanate (TRITC), Texas red,cyanine dyes (Cy3 and Cy5, for example), and the like; and enzymes thatreact with calorimetric substrates such as horseradish peroxidase. Theuse of fluorescent dyes is generally preferred in the practice of theinvention as they can be detected at very low amounts. Furthermore, inthe case where multiple antigens are reacted with a single array, eachantigen can be labeled with a distinct fluorescent compound forsimultaneous detection. Labeled spots on the array are detected using afluorimeter, the presence of a signal indicating an antigen bound to aspecific antibody.

The formation of antibody:antigen complexes can be performed under avariety of conditions to identify antibodies with varying bindingcharacteristics. Antigen-containing reaction solutions can containvarying degrees of salt or be conducted at varying pH levels. Inaddition, the binding reaction can be carried out at varyingtemperatures. Each set of conditions will identify antibodies withdifferent affinity for the antigen. For example, antibodies that bind atpH 2 may have utility under highly acidic conditions such as those thatexist in the stomach. Similarly, antibodies that bind at temperaturesnear boiling may be useful in studying thermophilic organisms. Ingeneral pH conditions will range from 2-10 (most preferably around pH8), temperatures from 0° C.-100° C. and salt conditions from 1 μM to 5 M(in the case of NaCl).

Affinity constants are a measure of the interaction between a particularligand and its cognate receptor. The “binding affinity” or the measureof the strength of association between a particular antibody:antigeninteraction is generally measured by affinity constants for theequilibrium concentrations of associated and dissociated configurationsof the antibody and its antigen. Preferably the binding of the antigenshould occur at an affinity of about k_(a)=10⁻⁶ M or greater to beuseful for the present invention, with greater than about 10⁻⁷ M beingmore preferable, and most preferably between about 10⁻⁸ M and about10⁻¹¹ M. Antibody fragments will generally have affinities in the rangeof about 10⁻⁶ M to 10⁻⁷ M.

In another embodiment of the invention, microarrays of uncharacterizedantibodies are used to compare the protein expression profiles of cells.For example, comparisons can be made between a population of cells fromone tissue, such as arterial endothelial cells, and a second tissue,such as venous endothelial cells or from cells derived from a particulartissue but from different species. Comparisons can be made betweennormal cells and cells from the same tissue type that originate from anindividual with a pathogenic disorder. For example, comparisons can bemade between normal cells and cancer cells. Comparisons can additionallybe made between cells in a resting state and cells in an activatedstate, for example, resting T-cells and activated T-cells.

In another example, the disclosed arrays are useful for evaluating theexpression of proteins by pathogens, such as, for example, bacteria,parasites, viruses, and the like. A solution (such as a lysate) madefrom the pathogen which represents all proteins expressed by thepathogen can be used to contact an antibody array to identify antibodiesrecognizing pathogen-expressed proteins. These antibodies have utilityas diagnostic agents as well as potential therapeutics.

Cellular lysates can be used as “antigens” as described above andreacted with two identical microarrays. Antibodies reactive in one arraybut not the other would indicate the presence of a differentiallyexpressed protein. This antibody is then useful for the subsequentisolation and identification of those proteins that are different in twopopulations of cells. In the case of normal and cancer cells, forexample, one may be able to identify proteins expressed in the cancercell that contribute to its malignant state.

In a further aspect of the invention, microarrays can be composed ofpreviously characterized antibodies. These microarrays have a variety ofuses, one of which is cell profiling. For example, an array can becomposed of antibodies that recognize a set of antigens known to bepresent in activated T-cells but not in resting T-cells. A population ofT-cells can then be lysed and the lysate contacted with the array todetermine if the population has the profile of activated or restingT-cells.

Microarrays and the methods disclosed herein can be used in methods ofdiagnosing particular disorders. For example, a collection of antibodiesspecific for a range of antigens associated with one or more disorderscan be arrayed and contacted with a bodily fluid containing antigenswhose presence, or absence, would indicate a particular disorder. Theadvantage of using a microarray over a conventional immunoassay is theability to include a population of antibodies diagnostic for a varietyof disorders on a single surface, significantly reducing time, costs andmaterials needed to effect a diagnosis.

For example, if a patient presents with symptoms that are characteristicof several distinct disorders which can be distinguished on the basis ofthe presence or absence of one or more proteins, a single microarrayassay could be used to make a specific diagnosis, thus allowing thepatient to be properly treated. Patients suffering from stroke or braininfarcts release several proteins into cerebrospinal fluid, examples ofwhich are neuron specific enolyse (NSE) from neuronal cells and S-100from glial cells and astrocytes. Such proteins are not released inconditions that may have similar symptoms, such as drug reactions,making proper diagnosis more difficult. A diagnostic array could readilydetect these and other proteins in the CSF, leading to a rapid clinicaldiagnosis and treatment.

In another aspect of the invention microarrays are employed tocharacterize protein expression patterns using nucleic acid samples.Briefly, nucleic acid molecules from a whole cell or tissue are appliedto a solid support using a microarray format. The arrayed nucleic acidsamples are then contacted with a nucleic acid probe specific for a geneencoding a known protein. The probe solution is left in contact with thearray for an amount of time sufficient to allow sample:probe complexesto form, then the unbound probe is washed away under suitable conditions(see, for example, Ausubel, et al, Short Protocols in Molecular Biology,3rd ed. 1995 and the examples below). Bound probe is detected at one ormore nucleic acid sample spots using one of a variety of detectionmethods.

This aspect of the invention has a variety of uses. For example, themicroarray can be constructed from nucleic acid samples isolated from asingle tissue type but from a large number of species, with each spotrepresenting a particular species. Thus in a single assay format one candetermine the evolutionary development of the protein represented by theprobe. Similarly, the microarray can be constructed of multiple tissuetypes from a single species, or from different developmental stages of asingle species (or multiple species) thus simply and efficientlydetermining tissue expression of the protein represented by the probe.For example, a microarray can be constructed with arrayed samplesrepresenting all the developmental stages of Drosophila, a well knownorganism the study of which has led to a greater understanding ofmammalian physiology and development.

The nucleic acid sample can be messenger ribonucleic acid (mRNA) or canbe complementary deoxyribonucleic acid (cDNA), including EST fragments.Methods for extracting and isolating nucleic acids from cells are wellknown in the art (for example phenol extraction/ethanol precipitation,ammonium acetate precipitation, cesium chloride gradients, and thelike), as are methods for generating cDNA (see, for example, “MolecularCloning: A Laboratory Manual,” second edition, edited by Sambrook,Fritsch, & Maniatis, Cold Spring Harbor Laboratory, 1989; and Ausubel,et al, Short Protocols in Molecular Biology, 3rd ed. 1995, both of whichare incorporated by reference herein). Microarrays of these nucleicacids are created using the methods described above. Techniques forcoupling nucleic acids to solid supports used to construct microarraysare well known in the art, including the poly-L-lysine and phenylboronicacid methods described in the Examples below.

The nucleic acid probes used in the invention methods can be designedbased on the sequence of a gene encoding a known protein or can be anEST fragment, as described above. One skilled in the art can readilydesign such probes based on the known sequence using methods of computeralignment and sequence analysis known in the art (e.g., “MolecularCloning: A Laboratory Manual”, second edition, edited by Sambrook,Fritsch, & Maniatis, Cold Spring Harbor Laboratory, 1989; Ausubel, etal, Short Protocols in Molecular Biology, 3rd ed. 1995). The probe cancomprise any number of nucleotides but will preferably be not fewer than10 nucleotides and preferably not more than about 300 nucleotides inlength.

The probes of the invention can be labeled by standard labelingtechniques such as with a radiolabel, enzyme label, fluorescent label,biotin-avidin label, chemiluminescent label, and the like. Afterhybridization, the probes may be detected using known methods. Preferredlabels are fluorescent labels, as described above.

The nucleic acid probes of the present invention include RNA as well asDNA probes and nucleic acids modified in the sugar, phosphate or eventhe base portion as long as the probe still retains the ability tospecifically hybridize under conditions as disclosed herein. Such probesare generated using techniques known in the art.

The term “hybridize” as used herein refers to a method of interacting anucleic acid sequence with a DNA or RNA molecule in solution or on asolid support, such as cellulose or nitrocellulose. If a nucleic acidsequence binds to the DNA or RNA molecule with sufficiently highaffinity, it is said to “hybridize” to the DNA or RNA molecule. Thestrength of the interaction between the probing sequence and its targetcan be assessed by varying the stringency of the hybridizationconditions. Various low to high stringency hybridization conditions maybe used depending upon the specificity and selectivity desired.Stringency is controlled by varying salt or denaturant concentrations.Examples of hybridization conditions are shown in the Examples below.Those skilled in the art readily recognize how such conditions can bevaried to vary specificity and selectivity. For example, under highlystringent hybridization conditions only highly complementary nucleicacid sequences hybridize. Preferably, such conditions preventhybridization of nucleic acids having even one or two mismatches out of20 contiguous nucleotides.

In a further aspect of the invention, microarrays can be composed ofrandomly generated polynucleotides (DNA or RNA) and contacted withproteins to identify unique binding pairs. Polynucleotides are now knownto bind to proteins and may have potential as diagnostics andtherapeutics (see, for example, Allen, et al, Virology 209(2):327-336,1995; Binkley, et al, Nucleic Acids Res. 23(16):3198-3205, 1995).Polynucleotides can be evaluated in very large numbers using the methodsdisclosed herein thus increasing the likelihood of identifying a usefulbinder.

The invention will now be described in greater detail by reference tothe following non-limiting examples.

EXAMPLES Example 1 Nucleic Acid Microarrays

The following procedures are conducted at room temperature and usingdouble distilled water unless otherwise noted. These methods areapplicable to arrays of polypeptides or polynucleic acids.

Glass slides are prepared as follows: NaOH (50 g) is dissolved in 150 mlof double distilled water (ddH₂O), then 200 ml of 95% EtOH is addedwhile stirring. If the solution becomes cloudy, ddH₂O is added until itbecomes clear. Approximately 30 glass slides (Gold Seal, Cat. No. 3010)are soaked in the NaOH/EtOH solution for 2 hours, shaking. The slidesare then rinsed three times with ddH₂O. The slides are next soaked in apoly-L-lysine solution (70 ml poly-L-lysine (Sigma Cat. No. 8920) to 280ddH₂O) for 1 hour. Excess liquid is removed by spinning the slides in arack on a microtiter plate carrier at 500 rpm. The slides are dried at40° C. for 5 minutes, then stored in a closed box for at least 2 weeksprior to use.

A cDNA microarray is prepared as follows: Total mRNA is isolated fromtissue (for example, nerve cells) of a variety of species representativeof different classes of organisms such as Drosophila, nematode, salmon,clam, chicken, mouse, dog, goat, spider monkey, chimpanzee, human, andthe like, by the FastTrac method (Stratagene, La Jolla, Calif.) or othercommon methods. mRNA is also obtained from a variety of unicellularorganisms such as E. coli, yeast, B. subtilis, mycoplasma and the like.Eukaryotic mRNA is enriched from total RNA using oligo(dT) cellulose(Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed. 1995, pgs4-11-4-12). Equivalent amounts (for example, 1 μg) of mRNA from eachsource are placed in a separate well of one or more 96 well microtiterplates and precipitated with cold EtOH. The precipitate is rinsed with70% EtOH and allowed to dry.

The dried mRNA is resuspended in 3×SSC (sodium chloride/sodiumcitrate-20× solution is 3 M NaCl (175 g/L0 0.3 M trisodium citrate 2H₂O(88 g/L adjusted to pH 7.0 with 1 M HCl) then spotted onto a previouslyprepared glass slide using an array device (for example, Shalon andBrown (WO 95/35505, published Dec. 28, 1995)). The prepared array can bekept for a long period of time before probing, however, if the slidesare to be kept for long periods of time, stability is increased byconverting each mRNA sample into cDNA using techniques known in the art,such as PCR.

The array is rehydrated by suspending the slide over a dish of ddH₂O(50° C.) for approximately one minute. The slide is quickly(approximately 3 seconds) dried by placing it on a surface heated to100° C. (mRNA side up). The mRNA is crosslinked to the poly-L-lysinecoating of the slide using ultraviolet radiation using a Stratalinker™UV device according to the manufacturer's instructions (Stratagene) setat 60 milliJoules.

The slides are next soaked in a solution of 5 grams of succinicanhydride (Aldrich Cat. No. 23, 969-0) dissolved in 315 ml ofN-methyl-pyrrilidinone (Aldrich Cat. No. 32,963-4) plus 35 mls of 0.2 Msodium borate (brought to pH 8.0 with NaOH) for 15 minutes with shaking.The slide is then transferred to a 95° C. water bath for 2 minutesfollowed by 95% EtOH for 1 minute. Excess liquid is removed from theslides by spinning a rack of slides on a microtiter plate carrier at 500rpm.

A probe sequence of a known protein (for example, human nerve growthfactor, GenBank Accession No. E03589) is labeled using standardprotocols, for example by using a CyDye™ Nick Translation kit(Amersham). The labeled probe (approximately 1 μg/ml) is resuspended in4×SSC (10 μl) to which is added 0.2 μl 10% sodium dodecyl sulfate (SDS).The probe is boiled for 2 minutes, then cooled for 10 seconds andtransferred to the array by pipette. The array is covered by a 22 mm×22mm cover slip, and the slide is placed in a humid hybridization chamberand submerged into a hot water bath (>75° C.).

The slide is left in the bath for 10-24 hours, then the cover slip isremoved and the slide rinsed in 0.2×SSC with 0.1% SDS several times.Excess wash buffer is removed by centrifugation on a microtiter platecarrier as described above. The slide is scanned using aspectrofluorometer, such as the ScanArray 3000 (General Scanning Inc.,Watertown, Mass.). For probes labeled with Cy5, for example,fluorescence is measured at 670 nm. Localization of spots on the arrayto which the probe hybridizes indicates that the species represented bythe spot expresses a protein similar or identical to the probe protein.

The procedure outlined below is an alternative method for bindingarrayed molecules to a solid support, using an SA(OCH₂CN)—X—NHS linkage(see, for example, U.S. Pat. No. 5,594,111, issued Jan. 14, 1997; U.S.Pat. No. 5,648,470, issued May 15, 1997; U.S. Pat. No. 5,623,055, issuedApr. 22, 1997; all of which are incorporated by reference herein).

Glass slides (Fisher Catalog No. 12-544-4) are soaked in an acid bath (1hour in 0.1 M HCl), then washed with water and dried at roomtemperature. The slides should not be aggressively dried, such as in anoven. The slides are next soaked in a silane solution overnight at roomtemperature (5% APTES (3-aminopropyl-triethoxysilane, Aldrich 28,177-8), 0.3% DIEA (Sigma) v/v in EtOH). The slides may be sonicated for10-15 minutes right after being placed in the APTES solution.

The slides are rinsed with isopropyl alcohol, then sonicated inisopropyl alcohol for several minutes. Sonication should remove anywhite silane residue on the slides. If the residue remains, the slidesshould be discarded. After sonication, the slides are left to cure/dryfor at least 24 hours before use.

The cured slides are next soaked in a linker solution overnight at roomtemperature. The linker solution is made by dissolving 115 mg of 9YSA(OCH₂CN)—X—COOH (Prolix, Bothell, Wash.) in 1 ml dimethylformamide(DMF) plus 60 μl DIEA, then adding 60 mg TSTU (Sigma) and leaving for 15minutes at room temperature. This stock is diluted in 270 ml ofisopropyl alcohol plus 270 μl DIEA before using.

The slides are removed from the linker solution and soaked in 1 M NH₂OH,1 mM EDTA, 0.1 M NaHCO₃ (pH 10) for 4 hours at room temperature. Thissolution is removed, the slides are extensively washed with water thenlet air dry at room temperature. The slides can be stored at roomtemperature away from light before using to make arrays.

Example II Determination of Optimal Concentrations of Antibody andAntigen

Various concentrations (1 μg/μl, 100 ng/μl, 10 ng/μl, 1 ng/μl) of totalmouse IgG or a mouse monoclonal anti-PLC-gamma were spotted on aldehydeslides (Cel Associates, Inc., Houston, Tex.), which allow non-covalentattachment of proteins. Using a manual 8 pin hand arrayer the slideswere blocked for 1 hour with PBST (phosphate buffered saline and 0.10%Tween 20), and 3% milk protein. The slides were subsequently washedthree times, 15 minutes each, in PBST. Duplicate slides were incubatedwith 50 μl of goat anti-mouse IgG antibody (GAMG) conjugated with CY3 orCY5 fluorescent dye compounds (Amersham, Arlington Heights, Ill.) at 10μg/ml or 1 μg/ml. Slides were then washed for 15 minutes in PBST threeadditional times and dried by centrifugation prior to scanning. Bindingwas detected as shown in Table 1 below.

Example III Comparison of Solid Supports

Serial dilutions (1 μg/ml, 100 ng/ml, 10 ng/ml, 1 ng/ml) of mouse IgG orPLC-gamma were hand arrayed onto aldehyde, polystyrene, nitrocelluloseand Surmodics slides. Aldehyde, nitrocellulose, polystyrene andSurmodics slides were purchased from various outside vendors (aldehydeSlides-Cel Associates, Inc., Houston, Tex.; nitrocellulose Slides-Molecular Probes, Inc., Eugene, Oreg.; polystyrene Slides-Nunc, Inc.,Naperville, Ill.; Surmodics Slides-Surmodics, Inc., Eden Prairie,Minn.). Surnodics slides have an undisclosed polymer on the glasssurface which forms a covalent linkage with proteins under theappropriate conditions (described by the manufacturer).

Following hand arraying of the antibodies (approximately 20-30nanoliters per spot), the nitrocellulose, aldehyde, and polystyreneslides were immediately blocked for 1 hour with PBST and 3% milk, washed3 times with PBST, and hybridized with 50 μl of GAMG-CY3 for 30 minutes.Surmodics slides were incubated overnight in a moist salt chamber asrecommended by the manufacturer. The following day, the Surmodics slideswere processed as described above. Following hybridization all of thevarious slides were washed 3 times in PBST, dried and scanned using aScan Array 3000 fluorescent scanner.

TABLE 1 Detection Antibody Conc. Antigen Conc. Level PLC-gamma 1 μg/μlGAMG-CY3 10 μg/ml +++ 100 ng/μl 10 μg/ml +++ 10 ng/μl 10 μg/ml + 1 ng/μl10 μg/ml − mouse IgG 1 μg/μl GAMG-CY3 10 μg/ml +++ 100 ng/μl 10 μg/ml+++ 10 ng/μl 10 μg/ml + 1 ng/μl 10 μg/ml − PLC-gamma 1 μg/μl GAMG-CY3 1μg/ml + 100 ng/μl 1 μg/ml + 10 ng/μl 1 μg/ml − 1 ng/μl 1 μg/ml − mouseIgG 1 μg/μl GAMG-CY3 1 μg/ml + 100 ng/μl 1 μg/ml + 10 ng/μl 1 μg/ml − 1ng/μl 1 μg/ml − PLC-gamma 1 μg/μl GAMG-CY5 10 μg/ml +++ 100 ng/μl 10μg/ml +++ 10 ng/μl 10 μg/ml + 1 ng/μl 10 μg/ml − mouse IgG 1 μg/μlGAMG-CY5 10 μg/ml +++ 100 ng/μl 10 μg/ml +++ 10 ng/μl 10 μg/ml + 1 ng/μl10 μg/ml − PLC-gamma 1 μg/μl GAMG-CY5 1 μg/ml + 100 ng/μl 1 μg/ml + 10ng/μl 1 μg/ml − 1 ng/μl 1 μg/ml − mouse IgG 1 μg/μl GAMG-CY5 1 μg/ml +100 ng/μl 1 μg/ml + 10 ng/μl 1 μg/ml − 1 ng/μl 1 μg/ml − +++ strongsignal, ++ moderate signal + weak signal, − no signal

All of the slides tested allowed for the detection of antigen:antibodybinding at higher concentrations of antibody. The Aldehyde andNitrocellulose treated slides were the most efficient at bindingantibody, and antibody:antigen interaction could be detected at 1 ng/μl.

Example IV Detection of Binding Using Non-Fluorescent Methods

Positively charged nylon filters (Zeta Probe Membranes, BioRadLaboratories, Hercules, Calif.) were hand arrayed using 1 μl ofanti-His, anti-V5, anti-thioredoxin (anti-Thio), anti-FOS,anti-PLC-gamma and anti-CREB antibodies (Invitrogen, Carlsbad, Calif.;all antibodies were approximately 1 mg/ml). Filters were blocked for 1hour with PBST and 3% milk, washed three times with PBST, and incubatedwith 1 μg/ml biotinylated D1 protein for three hours at roomtemperature. D1 is a creatine kinase fusion protein isolated from ahuman fetal heart cDNA library and cloned into the pBAD-Thio-His-TOPOvector (Invitrogen, Carlsbad, Calif.) to create aThioredoxin-V5-His-creatine kinase fusion protein. D1 was biotinylatedusing the EZ-Link™ Sulfo-NHS-LC Biotinylation Kit (Pierce, Rockford,Ill.) used according to the manufacturer's instructions).

Following three additional washes with the same buffer, filters weretreated with streptavidin/alkaline phosphatase conjugate orstreptavidin/horseradish peroxidase conjugate (Boehringer Mannheim, GmbHGermany) for 1 hour at room temperature.

The filters were washed 5 times with PBST, dried, and developed byimmersion in ECL chemiluminescent substrate (ECL-Amersham, ArlingtonHeights, Ill.) or the chromogenic substrate BCIP/NBT (Sigma Chemicals,St. Louis, Mo.). Filters developed with ECL were exposed to Kodakchemiluminescent film for 1 to 10 seconds.

The results are shown in FIGS. 1A, 1B, and 1C. In all cases, only theantibodies specific for epitopes on the fusion protein antigen weredetectable, and only in the arrayed spots, showing that the system hasboth good signal to noise ratio and specificity.

The experiment was repeated using an array created with an automatedarrayer. Antibodies (1 mg/ml) were spotted using an automated 96 pinmicroarrayer developed at Invitrogen. Fifteen negative controlantibodies (assorted mouse monoclonals) were arrayed along with thethree positive control antibodies (anti-His, anti-Thio, anti-V5).Filters were treated as described above using the alkaline phosphataseconjugate and the chromogenic substrate BCIP/NBT.

As can been seen in FIG. 2, binding and detection of antibody:antigencomplexes was highly specific and sensitive.

Example V Evaluation of Antibody Affinity

Anti-His, anti-V5, anti-FOS, anti-PLC-gamma, 25C1DG, and anti-VEGF(vascular endothelial growth factor) antibodies were arrayed on anitrocellulose slide and reacted with biotinylated D1 protein aspreviously described. Binding was detected with streptavidin-Cy3 asdescribed above. The anti-V5 antibodies spots showed red, the anti-Hisspots showed green, while the negative controls were undetectable (seeFIG. 3). When viewed in a black and white drawing, relative increase inbinding affinity is visualized by an increase of white in a given area.The color of the spots generally indicates a higher amount offluorescently labeled antigen present, and thus indicates relativebinding affinity between antibody and antigen. Colors, in descendingorder from highest to lowest affinity, are white, red, yellow, green,and blue. Using this technique, multiple antibodies can be tested fortheir affinity to a single antigen.

Example VI Polyclonal Antibody Microarrays

To demonstrate specific binding to polyclonal antibodies, six antibodieswere arrayed by hand on a nitrocellulose slide, three polyclonalantibodies (anti-E12 (unpurified rabbit polyclonal sera to aHis-V5-thioredoxin-thymidine kinase fusion protein), anti-lexA (lexArepressor protein), and anti-GFP(Green fluorescent protein)) and threemonoclonal antibodies (anti-V5, anti-His and anti-GalU (a mammaliantranscription factor). The slide was blocked with PBST and 3% milk for 1hour at room temperature, and incubated with the E12-biotin conjugate,prepared according to the protocol used for D1 protein. Followingextensive washing with PBST, the slides were incubated withstreptavidin-CY3 conjugate (Amersham, Arlington Heights, Ill.) for 1hour at room temperature, washed 5 times with PBST and dried bycentrifugation prior to scanning on the Scan Array 3000.

As can be seen in FIG. 4, binding was detected with both the antigenspecific polyclonal antibody (anti-E12) and the antigen specificmonoclonal antibodies (anti-His, anti-V5) and not with any of thenegative control antibodies.

Example VII Microarray Analysis of Labeled Cell Lysate

A series of experiments were conducted to determine if a microarray ofantibodies could specifically detect antigens in a cell lysate.

CHO cells expressing high levels of beta-galactosidase were grown toconfluency in a T-175 flask. (Hams media with Pen/Strep, and L-glutamineplus 10% FCS, at 37° C. with 5% CO₂) Cells were harvested usingTrypsin/EDTA. NP40 extracts were prepared by pelleting the cells (10⁷cells), washing once in PBS and resuspending in 5% NP40. Cell debris wasremoved by centrifugation. Soluble protein was biotinylated using aPierce biotinylation kit according to the manufacturer's instructions.

Nitrocellulose slides (see above) containing arrayed monoclonalantibodies (anti-beta-gal, anti-His, anti-Thio, anti-V5, anti-FOS,anti-PLC-gamma, anti-VEGF and 25C 10G (an anti-CREB antibody) wereblocked, washed, hybridized and developed with streptavidin-CY3 asdescribed in Example VI supra. As can be seen in FIG. 5A,beta-galactosidase binding was seen, however, some non-specific bindingwas detected as well.

The experiment was repeated, except that after centrifugation of theextract, soluble protein was dialyzed overnight against 50 mM phosphatebuffer at 4° C. prior to biotinylation. As can be seen in FIG. 5B, muchof the non-specific binding seen in the previous experiment waseliminated.

In the next experiment dialyzed extract containing the biotinylatedsoluble proteins was adjusted to 10% glycerol to reduce non-specifichydrophobic interactions. Furthermore, the sodium chloride concentrationwas adjusted to 0.2 M NaCl to increase specific ionic interactions. Allother conditions remained identical. As can be seen in FIG. 5C, allnon-specific binding was eliminated using this protocol.

While the foregoing has been presented with reference to particularembodiments of the invention, it will be appreciated by those skilled inthe art that changes in these embodiments may be made without departingfrom the principles and spirit of the invention, the scope of which isdefined by the appended claims.

1-50. (canceled)
 51. A method of comparing protein expression in two ormore populations of cells, said method comprising: (a) contacting anarray of antibodies bound to a solid surface with a cell lysate of afirst cell population, generating a first binding pattern; (b)contacting a duplicate array of antibodies on a solid surface with acell lysate of a second cell population, generating a second bindingpattern; and (c) comparing the binding pattern of the first cell lysatewith the binding pattern of the second cell lysate.
 52. The methodaccording to claim 51, wherein the antibodies are of uncharacterizedbinding specificity.
 53. The method according to claim 51, wherein theantibodies are recombinant antibodies.
 54. The according to claim 51,wherein the first cell lysate is derived from normal cells and thesecond cell lysate is derived from abnormal cells.
 55. The methodaccording to claim 54, wherein the abnormal cells are cancer cells. 56.The method according to claim 51, wherein the first cell lysate isderived from cells in a resting state and the second cell lysate isderived from cells in a stimulated state.
 57. The method according toclaim 51, wherein at least one of the one of the two cell lysatescomprises a detectable label.
 58. The method according to claim 51,wherein each of the two cell lysates comprises a different detectablelabel.
 59. A method of comparing protein expression in two or moreprotein samples, said method comprising: (a) contacting an array ofantibodies bound to a solid surface with a first protein sample,generating a first binding pattern; (b) contacting a duplicate array ofantibodies on a solid surface with a second protein sample, generating asecond binding pattern; and (c) comparing the binding pattern of thefirst protein sample with the binding pattern of the second proteinsample.
 60. The method according to claim 59, wherein the first andsecond protein samples are derived from a body fluid.
 61. The methodaccording to claim 59, wherein the first and second protein samples arederived from serum samples.
 62. The method according to claim 59,wherein the first and second protein samples are derived from cerebralspinal fluid, blood, plasma, urine, feces, saliva, tears, or extractedtissue.
 63. The method according to claim 59, wherein the first andsecond protein samples are derived from cellular extracts.
 64. A methodof diagnosing a disorder comprising: (a) Contacting an array ofantibodies specific for one or more antigens characteristic of adisorder with a biological sample obtained from a subject underconditions suitable for the formation of an antigen:antibody complex,wherein the presence of the antigens in the biological sample would beindicative of the disorder; and (b) Detecting the formation of anyantibody:antigen complexes.
 65. The method according to claim 64,wherein the biological sample is cerebral spinal fluid, blood, plasma,urine, feces, saliva, tears, or extracted tissue.
 66. The methodaccording to claim 64, wherein the disorder is stroke, cerebralhemorrhage, myocardial infarction, peripheral blood clots, diabetes,cancer, Alzheimer's disease or sepsis.